色谱分析前处理技术的新进展

目的：介绍色谱分析前处理技术的新进展。方法：将前处理技术大致分为样品制备技术和预处理、进样技术两大类，并分别查阅近斯的大量文献资料，筛选、整理各方法的原理、与其他方法比较的技术优势、应用领域等内容。结果：样品制备技术中的自动索氏提取、微波辅助溶剂萃取和加速溶剂萃取等3项技术主要适合于从中药等固态样品中彻底性地萃取待测总成分；预处理、进样技术中的固相萃取、固相微萃取、支持液膜萃取、微孔膜液液萃取、液相微萃取、电萃取、逆流分配和膜萃取等8项技术主要适合于从生物体液等液态样品中选择性地萃取待测成分，以备用于色谱进样。结论：许多分析工作者在色谱分析前处理技术领域内做了大量工作，并取得了进展。将来的趋势是发展少用有毒有机溶剂、简单快速便宜、适应特殊需求、能处理复杂介质中的痕量成分的方法，并发展的联用与自动化。只有克服前处理这一“瓶颈”技术，色谱分析才能实现真正意义上的飞跃。     

新型限进色谱在临床药物分析中的应用

限进填料属于特殊萃取介质之一,允许复杂生物基质样品直接进样分析,结合液相色谱系统可以实现生物样品自动化直接进样,简化并加速了样品预处理过程。本文简要介绍了限进填料固相萃取原理及分类,并对该技术在临床药物分析及药代动力学领域中的应用现状及发展趋势作了详细的综述。     

固相萃取技术及其在体内药物分析中的应用

目的了解固相萃取技术的新进展及其在体内药物分析中的应用情况。方法介绍固相萃取基本原理、填料种类和自动化操作等,并对该技术在体内药物分析中的应用进行综述。结果与结论固相萃取技术萃取回收率高、易于自动化,能有效去处样品中的杂质,适于体内药物分析中生物样品的预处理。     

固相微萃取技术及其在药物分析中的应用

固相微萃取(SPME)技术作为一种样品前处理方法，能够对样品中的痕量分析物进行富集，具有操作简单、高通量、有机溶剂用量少、易自动化的特点。该技术集提取、浓缩、进样于一体，大幅提高了萃取效率。该研究在介绍SPME技术的基础上，综述了近年来SPME在药物分析领域的应用，并简要探讨其局限性与发展前景，为复杂基质中的痕量组分检测提供了参考。     

固相萃取技术及其在体内药物分析中的应用

目的：了解固相萃取技术进展及应用情况。方法：从萃取机制、方法建立和萃取装置等对固相萃取技术进行综述，并对近年来该技术在体内药物分析方面的应用作了介绍。结果和结论：固相萃取技术萃取回收率高、易于自动化，能有效去除样品中的杂质等，适于生物样品的预处理。     

不同血样处理对高效液相色谱法测定血清丝裂霉素含量的影响

目的:考查不同的血样处理方法对血清中丝裂霉素(mitomycin C,MMC)含量的影响.方法:使用waters HPLC仪,配有限进填料(RAM)直接进样固定相,流动相:甲醇-水(10:90);流速:0.8 mL·min-1;紫外二极管阵列检测器,比较了血清样品不同处理方法的重现性、稳定性,并与血清直接进样进行比较.结果:不同的血清样品处理方法对MMC含量的影响不同,在萃取法中用氯仿-异丙醇较醋酸乙酯萃取率高,沉淀法用乙腈沉淀对MMC含量影响较小.直接进样中MMC与血清中的蛋白质有良好的分离.结论:在MMC的血清样品处理方法中,用RAM-HPLC法直接进样,样品不经前处理,简单快速,精密度好,但对一些取样量较大且含量较低的样品,萃取法为一种有效的方法.     

用胶束流动相和微径柱切换法测定体液中药物含量

测定体液中药物的含量往往由于样品中高分子蛋白质易在色谱柱中析出,导致柱效下降。通常药物在进样前都需经提取浓缩。这些步骤不仅费时费力,而且增加了分析误差。HPLC柱切换是一种广泛应用的自动进行样品净化和待测物浓缩的技术。该技术是先将样品直接注入预柱,     

反相高效液相色谱法测定大鼠血浆中姜黄素含量的方法改进

目的采用改进的萃取剂用于血浆的预处理,HPLC法测定姜黄素的血药浓度。方法以乙酸乙酯与二甲基亚砜（14：1,V/V）作为萃取溶剂,一次萃取血浆样品。色谱柱为XDB C18柱（150×4.6mm,5μm）,流动相为乙腈-5%冰乙酸（45：55）,流速1.0mL.min-1,柱温30℃,检测波长428nm,进样量10.0μL。结果采用新型萃取剂处理血浆样品,萃取回收率高,操作简单,姜黄素血药浓度在0.02～5.0μg.mL-1范围内与峰面积的线性关系良好,回归方程Y=111.71X-3.1788,r=0.9998,日内、日间精密度RSD符合生物样品的分析要求。结论本方法简便、快捷、准确、重现性好,可用于姜黄素血药浓度测定及药代动力学研究。     

固相微萃取技术及其在药物分析中的应用

固相微萃取（SPME）是基于固相萃取（SPE）技术发展起来的,为一种集萃取、浓缩、解吸、进样等功能于一体的样品前处理方法。本文简要介绍了SPME技术的历史、工作原理、萃取装置及萃取方法等方面内容,并结合萃取方法,介绍了SPME技术在溶剂残留、体内药物分析、中药分析等药物分析相关领域中的应用。     

药物代谢转化和样品前处理技术的研究进展

本文对近年来药物代谢转化研究方法和样品前处理技术进行了综述。介绍了药物体内和体外代谢研究模型,以及代谢产物的分离和鉴定技术新进展,重点对固相萃取和固相微萃取的原理、提取步骤、各种实验参数的影响、与分析仪器联用技术的研究进展和应用进行了评述。     

The use of on-line and off-line chromatographic extraction techniques coupled with mass spectrometry for support of in vivo and in vitro assays in drug discovery

We have investigated various sample chromatographic extraction and sample preparation methods for liquid chromatography mass spectrometry analysis in order to increase the throughput of various in vivo and in vitro assays in support of drug discovery. The results indicated that direct plasma injection, although certainly faster than conventional protein precipitation for sample preparation, had problems associated with column longevity and overall robustness. Frequently a single study could not be completed without column replacement. On-line solid phase extraction, on the other hand, compared well with off-line solid phase extraction, using our LC extraction column design, as contamination of the extraction column was minimized by back flushing using the Gilson syringe pump. Finally, on-line solid phase extraction for support of Caco-2 permeability studies worked very well for both single components and mixtures as the matrix was much simpler, presenting fewer contamination problems.     

Recent advances in column switching sample preparation in bioanalysis

Column switching techniques, using two or more stationary phase columns, are useful for trace enrichment and online automated sample preparation. Target fractions from the first column are transferred online to a second column with different properties for further separation. Column switching techniques can be used to determine the analytes in a complex matrix by direct sample injection or by simple sample treatment. Online column switching sample preparation is usually performed in combination with HPLC or capillary electrophoresis. SPE or turbulent flow chromatography using a cartridge column and in-tube solid-phase microextraction using a capillary column have been developed for convenient column switching sample preparation. Furthermore, various micro-/nano-sample preparation devices using new polymer-coating materials have been developed to improve extraction efficiency. This review describes current developments and future trends in novel column switching sample preparation in bioanalysis, focusing on innovative column switching techniques using new extraction devices and materials.     

Direct injection analysis of melphalan in plasma using column-switching high-performance liquid chromatography.

An automated column-switching high-performance liquid chromatographic (HPLC) method with ultraviolet detection is described for a simple and rapid determination of melphalan, an alkylating agent, in human plasma following direct sample injection. The system consists of a pretreatment column and an analytical column connected in series via a switching valve. Plasma samples are loaded onto a pretreatment column with an aqueous mobile phase, with which the sample to be analyzed is retained and the solubilized plasma proteins are flushed to be discarded. The retained compound is eluted from the pretreatment column onto the analytical column by using the chromatographic mobile phase with a higher elution capacity. The column-switching technique can be used to achieve an automated assay. Analytical recovery of the compound was 99.1%, and the coefficients of both intra- and interday variations did not exceed 3.5%. The detection limit was 10 ng/ml for the compound. Recovery of melphalan in plasma was only 2.1% after 4 weeks at 25 degrees C, as compared to 24.5% at 5 degrees C and 100.1% at -20 degrees C.     

Bioanalysis of the enantiomers of (+/-)-sarin using automated thermal cold-trap injection combined with two-dimensional gas chromatography

A fully automated multidimensional gas chromatographic system with thermal desorption injection and alkali flame detection was developed for analysis of the enantiomers of the nerve agent (+/-)-sarin. The chiral stationary phase was CP Cyclodex B on which the sarin enantiomers were completely resolved. The absolute detection limit was 2.5 pg per enantiomer. The method is intended to be used for the analysis of the sarin enantiomers in biological samples. For this purpose, sarin was isolated from guinea pig blood via solid-phase extraction. Deuterated sarin was used as internal standard. Stabilization of sarin in the blood sample by acidification and addition of an excess of a competitive organophosphorus compound (neopentyl sarin) appeared to be essential. The absolute recovery of the extraction procedure was 60%, whereas the recovery relative to the internal standard was 100%.     

Direct determination of stability of protease inhibitors in plasma by HPLC with automated column-switching

Automated column-switching HPLC methods were developed and utilized for the direct analyses of three hydroxamic acid based metalloprotease inhibitors in rat plasma. These column-switching methods involved the use of a restricted-access media (RAM) precolumn and a column-switching valve, allowing the complete automation of sample preparation and HPLC. The plasma samples were directly injected onto a precolumn packed with SPS/ODS stationary phase and then backflushed onto an ODS analytical column using a 6-port column-switching device. The drug stability in rat plasma was determined using both the automated and traditional HPLC methods. The results obtained from the automated column-switching methods were in good agreement with those from traditional methods that involve sequential protein precipitation, liquid extraction, solvent evaporation, and sample reconstitution. In addition to the elimination of labor-intensive and time-consuming sample preparation procedures, the column-switching methods allowed on-line analyte enrichment and accurate determination of drug stability in plasma with detection limits in the range of 10-20 ng/ml(-1). This work represents, for the first time, a drug stability study in plasma by automated column-switching HPLC technique with the use of a RAM column. Our column-switching methods can be readily adapted to any existing HPLC system with minimal hardware modification.     

A method for the direct analysis of drug compounds in plasma using a single restricted access material (RAM) column

We describe an automated approach to analyzing whole plasma samples using online extraction without the need for an analytical column. A single restricted access material (RAM) column provided online extraction and pre-concentration of analytes while effectively removing proteins, salts and other biological materials found in the plasma sample matrix. The reduction in the plasma matrix enabled direct elution of the analytes from the extraction column to the mass spectrometer for selective detection. The precision of the method was evaluated using a proprietary therapeutic agent (Compound A) and was less than 5% over the range of 1-500ng/ml in spiked whole plasma, with an LOQ of 1ng/ml. A side-by-side comparison of RAM results from a pharmacokinetic study in rats was made with a traditional protein precipitation LC-MS method and a correlation of 0.993 was obtained between both methods. The injection-to-injection cycle time for the RAM method was 8min. Further automation was demonstrated by addition and mixing of the internal standard to all samples via an injection program of the autosampler.     

On-column electroextraction and separation of antisense oligonucleotides in human plasma by capillary gel electrophoresis

A novel approach is presented for the direct injection, and subsequent separation, of antisense phosphorothioate oligonucleotides in human plasma by capillary gel electrophoresis. The plasma, spiked with the antisense, was simply diluted 1:1 with acidified water and inserted into the sample holder in the capillary electrophoresis instrument. The separation capillary, filled with a dextran solution (replaceable polymer) and a short zone of acidified water at the injection side, was dipped into the plasma sample vial and voltage applied for simultaneous electrokinetic extraction and injection of antisense. The sample vial was then exchanged for the buffer vial, separation voltage applied, and size-sieving separation achieved. Separation time is less than 9 min and total time per analysis cycle 20 min, including rinsing of the capillary, filling with polymer, electroextraction/injection, and separation. This automated method can handle small sample volumes (4 microl) and has a detection limit of 0.5 microgml(-1) for a 16-mer phosphorothioate employing UV-detection. The capillary is stable for about 50 analyses.     

Improved method for morphine determination in biological fluids and tissues: rapid, sensitive and selective.

Morphine was assayed using a simple two step solvent extraction--acid back extraction sample preparation method, coupled with normal phase high-performance liquid chromatography (HPLC) and dual electrode coulometric detection. HPLC is performed with a 1.0 M Tris-methanol (5:95) mobile phase with subtle pH adjustments to separate morphine and internal standard from any interfering compounds. The use of normal phase HPLC (silica column) substantially reduces problems from interfering lipophilic substances sometimes encountered with reverse phase HPLC following solvent extraction and which would otherwise require more time-consuming sample preparation. Dual electrode detection further improves the selectivity for morphine and gives excellent sensitivity (0.5 ng mL-1), reproducibility and stability for automated sample injection. This method has proven suitable for pharmacokinetic studies of morphine.     

Rapid bioanalytical determination of dextromethorphan in canine plasma by dilute-and-shoot preparation combined with one minute per sample LC-MS/MS analysis to optimize formulations for drug delivery

The determination of dextromethorphan in canine plasma is used to demonstrate the high throughput bioanalytical approach of automated dilute-and-shoot (DAS) sample preparation followed by a 1 min isocratic liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Dilute-and-shoot preparation is commonly used for the determination of drugs in several biological matrices such as urine and saliva, but is not typically used with plasma samples because the amount of protein present in plasma can lead to a variety of problems including column failure. As a result, plasma sample preparation usually removes protein by precipitation, extraction or filtration; however, the dilute-and-shoot approach solubilizes proteins throughout the chromatographic portion of the assay. The attributes of this approach are compared with a previously validated liquid/liquid extraction procedure for determination of dextromethorphan in plasma. Accuracy and precision of both methods are similar. The lower limit of quantitation (LLOQ) of the dilute-and-shoot approach is much higher at 2 ng/ml versus 5 pg/ml with the liquid/liquid extraction; however, the sample throughput of the preparation portion of the dilute-and-shoot approach is more than 50-fold greater. The ruggedness of the dilute-and-shoot method was thoroughly investigated because of the problems traditionally associated with the direct injection of diluted plasma onto an LC-MS/MS instrument. With the optimal conditions, greater than 1,000 injections of diluted plasma have been successfully performed on a single column in less than 19 h making this technique an excellent approach for the rapid preparation and high throughput of plasma samples containing drug levels in the ng/ml range or higher. Application of this methodology to measure the levels of dextromethorphan in canine plasma to evaluate drug delivery from various formulations is also presented.